Proteomic characterization of endogenous substrates of mammalian ubiquitin ligase Hrd1
Abstract Background Endoplasmic reticulum (ER)-associated degradation (ERAD) regulates protein homeostasis in the secretory pathway by targeting misfolded or unassembled proteins for degradation by the proteasome. Hrd1 is a conserved multi-spanning membrane bound ubiquitin ligase required for ubiqui...
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| Format: | Article |
| Language: | English |
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BMC
2018-08-01
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| Series: | Cell & Bioscience |
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| Online Access: | http://link.springer.com/article/10.1186/s13578-018-0245-z |
| _version_ | 1819127855710208000 |
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| author | Yilin Ye Suk-Hwan Baek Yihong Ye Ting Zhang |
| author_facet | Yilin Ye Suk-Hwan Baek Yihong Ye Ting Zhang |
| author_sort | Yilin Ye |
| collection | DOAJ |
| description | Abstract Background Endoplasmic reticulum (ER)-associated degradation (ERAD) regulates protein homeostasis in the secretory pathway by targeting misfolded or unassembled proteins for degradation by the proteasome. Hrd1 is a conserved multi-spanning membrane bound ubiquitin ligase required for ubiquitination of many aberrant ER proteins, but few endogenous substrates of Hrd1 have been identified to date. Methods Using a SILAC-based quantitative proteomic approach combined with CRISPR-mediated gene silencing, we searched for endogenous physiological substrates of Hrd1. We used RNA microarray, immunoblotting, cycloheximide chase combined with chemical genetics to define the role of Hrd1 in regulating the stability of endogenous ERAD substrates. Results We identified 58 proteins whose levels are consistently upregulated in Hrd1 null HEK293 cells. Many of these proteins function in pathways involved in stress adaptation or immune surveillance. We validated OS9, a lectin required for ERAD of glycoproteins as a highly upregulated protein in Hrd1 deficient cells. Moreover, the abundance of OS9 is inversely correlated with Hrd1 level in clinical synovium samples isolated from osteoarthritis and rheumatoid arthritis patients. Intriguingly, immunoblotting detects two OS9 variants, both of which are upregulated when Hrd1 is inactivated. However, only one of these variants is subject to proteasome dependent degradation that requires Hrd1 and the AAA (ATPase associated with diverse cellular activities) ATPase p97. The stability of the other variant on the other hand is influenced by a lysosomal inhibitor. Conclusion Hrd1 regulates the stability of proteins involved in ER stress response and immune activation by both proteasome dependent and independent mechanisms. |
| first_indexed | 2024-12-22T08:18:34Z |
| format | Article |
| id | doaj.art-741fc29e166744a1a1d481da718963a0 |
| institution | Directory Open Access Journal |
| issn | 2045-3701 |
| language | English |
| last_indexed | 2024-12-22T08:18:34Z |
| publishDate | 2018-08-01 |
| publisher | BMC |
| record_format | Article |
| series | Cell & Bioscience |
| spelling | doaj.art-741fc29e166744a1a1d481da718963a02022-12-21T18:32:49ZengBMCCell & Bioscience2045-37012018-08-018111210.1186/s13578-018-0245-zProteomic characterization of endogenous substrates of mammalian ubiquitin ligase Hrd1Yilin Ye0Suk-Hwan Baek1Yihong Ye2Ting Zhang3Department of Orthopedics, Peking University First HospitalDepartment of Biochemistry & Molecular Biology, College of Medicine, Yeungnam UniversityLaboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of HealthLaboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of HealthAbstract Background Endoplasmic reticulum (ER)-associated degradation (ERAD) regulates protein homeostasis in the secretory pathway by targeting misfolded or unassembled proteins for degradation by the proteasome. Hrd1 is a conserved multi-spanning membrane bound ubiquitin ligase required for ubiquitination of many aberrant ER proteins, but few endogenous substrates of Hrd1 have been identified to date. Methods Using a SILAC-based quantitative proteomic approach combined with CRISPR-mediated gene silencing, we searched for endogenous physiological substrates of Hrd1. We used RNA microarray, immunoblotting, cycloheximide chase combined with chemical genetics to define the role of Hrd1 in regulating the stability of endogenous ERAD substrates. Results We identified 58 proteins whose levels are consistently upregulated in Hrd1 null HEK293 cells. Many of these proteins function in pathways involved in stress adaptation or immune surveillance. We validated OS9, a lectin required for ERAD of glycoproteins as a highly upregulated protein in Hrd1 deficient cells. Moreover, the abundance of OS9 is inversely correlated with Hrd1 level in clinical synovium samples isolated from osteoarthritis and rheumatoid arthritis patients. Intriguingly, immunoblotting detects two OS9 variants, both of which are upregulated when Hrd1 is inactivated. However, only one of these variants is subject to proteasome dependent degradation that requires Hrd1 and the AAA (ATPase associated with diverse cellular activities) ATPase p97. The stability of the other variant on the other hand is influenced by a lysosomal inhibitor. Conclusion Hrd1 regulates the stability of proteins involved in ER stress response and immune activation by both proteasome dependent and independent mechanisms.http://link.springer.com/article/10.1186/s13578-018-0245-zEndoplasmic reticulum-associated protein degradation/ERADProtein quality controlOS9ER stressERAD tuningLysosome degradation |
| spellingShingle | Yilin Ye Suk-Hwan Baek Yihong Ye Ting Zhang Proteomic characterization of endogenous substrates of mammalian ubiquitin ligase Hrd1 Cell & Bioscience Endoplasmic reticulum-associated protein degradation/ERAD Protein quality control OS9 ER stress ERAD tuning Lysosome degradation |
| title | Proteomic characterization of endogenous substrates of mammalian ubiquitin ligase Hrd1 |
| title_full | Proteomic characterization of endogenous substrates of mammalian ubiquitin ligase Hrd1 |
| title_fullStr | Proteomic characterization of endogenous substrates of mammalian ubiquitin ligase Hrd1 |
| title_full_unstemmed | Proteomic characterization of endogenous substrates of mammalian ubiquitin ligase Hrd1 |
| title_short | Proteomic characterization of endogenous substrates of mammalian ubiquitin ligase Hrd1 |
| title_sort | proteomic characterization of endogenous substrates of mammalian ubiquitin ligase hrd1 |
| topic | Endoplasmic reticulum-associated protein degradation/ERAD Protein quality control OS9 ER stress ERAD tuning Lysosome degradation |
| url | http://link.springer.com/article/10.1186/s13578-018-0245-z |
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